Automated enrichment for nucleic acid sequencing

ABSTRACT

The present teachings provide apparatuses and methods for automated handling of samples, e.g., biological or chemical samples. The apparatuses and the methods of the present teachings allow automated performance of various sample manipulation steps without manual intervention. In a preferred embodiment, the present teachings provide apparatuses and methods for automated enrichment of templated beads produced by PCR.

CROSS-REFERENCE TO RELATED APPLICATION(S)

This application claims benefit of U.S. Provisional Application No.61/505,770, filed Jul. 8, 2011, entitled “AUTOENRICHMEHT FOR NUCLEICACID SEQUENCING”, and naming inventor Kristopher Barbee, which isincorporated herein by reference in its entirety.

This application claims benefit of U.S. Provisional Application No.61/532,884, filed Sep. 9, 2011, entitled “METHOD AND APPARATUS FORAUTOMATED SAMPLE MANIPULATION”, and naming inventors Kristopher Barbeeand Maximilian Carpino, which is incorporated herein by reference in itsentirety.

This application claims benefit of U.S. Provisional Application No.61/532,903, filed Sep. 9, 2011, entitled “AUTOENRICHMEHT FOR NUCLEICACID SEQUENCING”, and naming inventors Kristopher Barbee, MaximilianCarpino, Ray Wheeling, and Mik Bajk, which is incorporated herein byreference in its entirety.

FIELD OF THE DISCLOSURE

The present teachings relate to an apparatus and method for automatedhandling of samples, e.g., biological or chemical samples, includingautomated performance of various sample manipulation steps withoutmanual intervention.

BACKGROUND

A number of biological sample analysis methods rely on samplepreparation steps as a precursor to carrying out the analysis methods.For example, a precursor to performing many biological sequencingtechniques (e.g., sequencing of nucleic acid) includes amplification ofnucleic acid templates in order to obtain a large number of copies(e.g., thousands or millions of copies) of the same template.

Polymerase chain reaction is a well understood technique for amplifyingnucleic acids which is routinely used to generate sufficiently large DNApopulations suitable for downstream analysis. Recently, PCR-basedmethods have been adapted to amplifying samples contained withinemulsions for sequencing applications. In such amplification methods aplurality of biological samples (e.g. nucleic acid samples) may bediscretely sequestered in microcapsules or droplets of an emulsion andPCR amplification conducted on each of the plurality of encapsulatednucleic acid samples simultaneously. Such microcapsules or droplets maybe referred to as “microreactors” because the amplification reactionoccurs within the microcapsule or droplet.

In some cases, the microreactor can include a template bead or particlewhich may serve as a support or carrier of amplified sample templates.The amplification process may be referred to as bead-based orparticle-based emulsion amplification, for example, as described in US2008/0003571 A1 to McKernan et al., which is incorporated herein in itsentirety by reference. In such a technique, beads or particles alongwith DNA templates are suspended in an aqueous reaction mixture and thenencapsulated in an inverse (water-in-oil) emulsion. The template DNA maybe either coupled to the bead or particle prior to emulsification or maybe included in solution in the amplification reaction mixture.

Emulsion amplification (e.g., ePCR) is a step in many next generationsequencing workflows. After ePCR is complete, the micro-reactors in theemulsion are broken, and the templated beads and nonamplifying beads arewashed to remove the oil and emulsifiers. Enrichment is performed toseparate templated beads from non-amplifying or poorly amplifying beads.

In a conventional enrichment step, polystyrene beads with asingle-stranded adaptor attached are used to capture templated beads.The mixture of enrichment beads, enrichment bead-templated beadcomplexes, and non-amplifying beads is centrifuged. The enrichment stepresults in a layer of enrichment beads (with or without templated beadsattached) at the top and a layer of non-amplifying beads at the bottom.The layer of enrichment beads is extracted and denatured to dissociatethe templated beads from the enrichment beads. The enrichment step isperformed manually, which is time consuming, with low yield andinconsistent quality of templated beads, and high cost.

It is therefore desirable to provide an automate enrichment process toenhance performance by minimizing complexity and cost of device andconsumables, reduce hands on time, increase yield, achieve higherpercentage of enriched particles, and minimize variability.

SUMMARY

The present teachings provide apparatuses and methods for automatedhandling of samples, e.g., biological or chemical samples. Theapparatuses and the methods of the present teachings allow automatedperformance of various sample manipulation steps without manualintervention. In a preferred embodiment, the present teachings provideapparatuses and methods for automated enrichment of templated beadsproduced by PCR.

BRIEF DESCRIPTION OF THE DRAWINGS

The present disclosure may be better understood, and its numerousfeatures and advantages made apparent to those skilled in the art byreferencing the accompanying drawings.

FIG. 1A is a perspective view of an automated sample handling system forconducting automated sample preparation according to various embodimentsof the present teachings.

FIG. 1B is an additional view of the device shown in FIG. 1A. This viewfeatures a slide block 7 in an OUT position.

FIG. 1C is a rear view of the device shown in FIG. 1A. This viewfeatures the holes 9 designed to accommodate a control rod 8.

FIG. 2 is a perspective view of an automated sample handling system forconducting automated sample preparation according to various embodimentsof the present teachings.

FIG. 3A is a perspective view of an automated sample handling system forconducting automated sample preparation according to various embodimentsof the present teachings.

FIG. 3B is an expanded view of the components of the system shown inFIG. 3A.

FIG. 3C shows a system having a receptacle for a container.

FIG. 4 is a perspective view of an automated sample handling system forconducting automated sample handling according to various embodiments ofthe present teachings.

FIG. 5 is a perspective view of an automated sample handling system forconducting automated sample handling according to various embodiments ofthe present teachings.

The use of the same reference symbols in different drawings indicatessimilar or identical items.

DETAILED DESCRIPTION

In various embodiments, the present teachings provide an apparatus forautomated sample manipulation including (a) a platform having aplurality of sites adapted to hold each of a plurality of samples ateach of said plurality of sites; (b) a magnet; (c) a mechanical stage orarm including a mount for a device having a compartment with an orifice,wherein the mechanical stage or arm is adapted to (c1) pick up thedevice; (c2) move the device to any of the plurality of sites and engagethe device with a sample at the site; and (c3) move one or more sampleson the platform via the device and a sample at an engaged site such thata sample is within a magnetic field of the magnet; (d) a pump connectingto the device; and (e) a control unit including a processer and memoryencoding one or more programs, wherein said control unit is adapted tocause the mechanical stage or arm or the pump to carry out one or moretasks.

In various embodiments, the present teachings provide an apparatus forautomated liquid sample manipulation, including (a) a platform having aplurality of sites adapted to hold each of a plurality of samples, asample at each of the plurality of sites; (b) at least one magnetmounted on a block; (c) a mechanical stage or arm including (c1) a mountfor a device having a compartment with an orifice; and (c2) a rod,wherein the mechanical stage or arm is adapted to (i) pick up thedevice, and move the device to any of the plurality of sites; and (ii)engage the rod to the block and move the magnet to or from one or moreof the plurality of sites; (d) a pump connecting to the device; and (e)a control unit including a processer and memory encoding one or moreprograms, wherein the control unit is adapted to cause the mechanicalstage or arm or the pump to carry out one or more tasks. The mechanicalstage or arm may be adapted to slide the block such that the magnet ismoved to or from a sample at a site at one end of the line of aplurality of sites.

In various embodiments, the present teachings provide an apparatus forautomated liquid sample manipulation, including (a) a platformcomprising a plurality of sites adapted to hold each of a plurality ofsamples at each of the plurality of sites; (b) at least one magnetmounted on a block, the block includes a rod; (c) a mechanical stage orarm comprising a mount for a device comprising a compartment having anorifice, wherein the mechanical stage or arm is adapted to (c1) pick upthe device, and move the device to any of the plurality of sites; and(c2) engage the rod on the block and move the magnet to or from one ormore of the plurality of sites; (d) a pump connecting to the device; and(e) a control unit including a processer and memory encoding one or moreprograms, wherein the control unit is adapted to cause the mechanicalstage or arm or the pump to carry out one or more tasks. The mechanicalstage or arm may be adapted to slide the block such that the magnet ismoved to or from a sample at a site at one end of the line of aplurality of sites.

The apparatus of the present teachings may further include a holder forholding the device. The device may be a pipette. The platform may be aline of a plurality of sites adapted to hold an multi-well strip. Theapparatus of the present teachings may comprise one magnet located atone end of the line of the plurality of sites. When the mechanical stageor arm moves the multi-well strip along the line, a sample at one end ofthe plurality of sites is within the magnetic field of the magnet. Theapparatus of present teachings may also comprise a first and a secondmagnet located at opposite ends of the line of the plurality of sites.When the mechanical stage or arm moves the multi-well strip along theline, a sample at one end of the plurality of sites is within themagnetic field of the first magnet or a sample at the opposite end ofthe plurality of sites is within the magnetic field of the secondmagnet. The apparatus of the present teachings may further include areceptacle for a container to which a processed sample may betransferred.

In various embodiments, the present teachings provide the method forautomated liquid sample manipulation, including (a) providing aplurality of samples at each of the plurality of sites, wherein each ofthe plurality of samples is held at one of the plurality of sites,wherein at least one sample of the plurality of samples comprises anonmagnetic portion and a magnetic portion; (b) transferring one or moresamples from one or more origin sites among the plurality of sites to asite holding the at least one sample comprising a nonmagnetic portionand a magnetic portion; (c) positioning the site holding the at leastone sample comprising a nonmagnetic portion and a magnetic portion in amagnetic field of the magnet; and (d) separating the nonmagnetic portionfrom the magnetic portion by removing the nonmagnetic portion from thesite holding the at least one sample comprising a nonmagnetic portionand a magnetic portion; wherein the steps (b)-(d) are performed withoutmanual intervention. The nonmagnetic portion may comprise a liquid andthe magnetic portion may comprise magnetic beads, and a separating stepinvolves immobilizing the magnetic beads by a magnet and removing theliquid by aspiration.

The method of the present teachings may also include a step of (e)treating at least one sample having a nonmagnetic portion and a magneticportion. The treating step may be any of degassing, mixing, heating, orstirring. The method may also include repeating the steps (b), (c), (d)or (e) in a predetermined sequence to effect separation of the templatedbeads from other components of the sample.

In various embodiments of the present teachings, the magnetic beads maybe templated beads produced in emulsion PCR. The magnetic beads may bemagnetic enrichment beads, complexes of magnetic enrichment bead andtemplated beads, and non-amplifying beads.

The present teachings apply to apparatuses and methods for automatedhandling of samples. The apparatuses and methods of the presentteachings allow automated performance of various sample manipulationsteps such as sample transfer, separation, and treatment, e.g.,degassing, mixing, heating, and stirring, without manual intervention.In a preferred embodiment, the present teachings provide apparatuses andmethods for automated enrichment of templated magnetic beads produced byPCR.

In various embodiments, the present teachings provide an apparatus whichmay comprise a holding means comprising a plurality of sites adapted tohold a plurality of samples each at one site, a transfer means fortransferring one or more samples from one or more origin sites among theplurality of sites to one or more destination sites among the pluralityof sites, a separation means for separating each of one or more samplesat one or more separation sites among the plurality of sites into aplurality of different fractions, a positioning means for positioningthe separation means and the samples at the separation sites by causingthe separation means and the samples at the separation sites to beengaged for separation or disengaged, and a control means forcontrolling the transfer means, the separation means, and thepositioning means by causing them to perform their respective functions.

In various embodiments of the apparatus, the holding means may be ahorizontal surface that may optionally be flat or shaped or slotted soas to securely accommodate the samples. For example, the holding meansmay be a device comprising a platform, a table, a tray or the like.

The number and arrangement of the sites is not particularly limited.Various embodiments may include a one or two-dimensional array with X byY number of sites where X corresponds to the number of samples to bemanipulated by one automated process. In a specific embodiment, X isone, and the array is a one-dimensional array, e.g., a strip. Thus, theY dimension corresponds to the number of sites for one samplemanipulation process, e.g., the number of reagents, solutions, buffers,washes, solvents, destination sites, waste reservoirs or various othermaterials needed in accordance with the procedure to be automated by theapparatus. Each of the materials used in the process are contained in asuitable container, e.g., a sample well, tube, cuvette or the like. Thesites/strips may or may not be attached to each other. The volume of thecontainer may vary along the Y dimension in accordance with the amountof material needed in various stages of the sample manipulation. Forexample, for each sample, there are a finite number of Y sites that mayeach have a unique volume in accordance with the prescriptions of aparticular automated manipulation procedure. In various embodiments, Ymay be at least 2, 4, 8, 16, 32, or 64. In another embodiment, the arrayis a two-dimensional array having a plurality of one-dimensional arrays.For example, X can be at least 2, 4, 8, 16, 32, or 64. Such embodimentsallow X automated manipulation processes to be carried out concurrentlyor in sequence.

The term “well” refers to a sample chamber or sample confinement area orregion, which can be a physical or chemical attribute of a substratethat permit the localization of a sample of interest. A well may be adiscrete region of a substrate. A well may be configured or associatedwith structural attributes such as hollows or wells having definedshapes and volumes which are manufactured into a substrate. The wellsmay have any suitable shape, such as square, rectangular, or octagonalcross sections, and may be arranged as a rectilinear array. Wells mayalso have hexagonal cross sections and be arranged as a hexagonal array,which permit a higher density of wells per unit area in comparison torectilinear arrays. In some embodiments, the array may contain a totalof 22, 23, 24, 25, 26 or 27. In other embodiments, the array of wellscomprises 102, 103, 104, 105, 106 or 107 wells. Wells can also be amicrotiter plate (“microplate”), which is a flat plate with multiplewells. The microplate can be a strip, e.g., an 8-well strip, or atwo-dimensional array, e.g., an array of 12, 24, 48, 96, 384 or 1536wells arranged in a X:Y rectangular matrix, e.g., a 2:3 rectangularmatrix.

The one or two dimensional tray may be preloaded with various materialsrequired for the sample manipulation procedure, for example, reagents,solutions, buffers, washes and solvents.

Various embodiments of the apparatus may include a transfer means fortransferring one or more samples from one or more origin sites to one ormore destination sites. An origin site is any site that contains asample that is then transferred. A destination site is any site that asample is transferred to. For example, a destination site may optionallybe a separation site, a capture site or a sample treatment site.Separation, capture and sample treatment may also occur at the samesite.

One example of a transfer means comprises a mechanical stage or arm thatis capable of moving in one or more directions (e.g., a robotic stage orarm) which may optionally be detachable. The mechanical stage or arm maybe capable of lateral and transverse motion so that all positions in atwo dimensional array may be accessed. The mechanical stage or arm mayalso be capable of vertical motion, i.e., up and down. Alternatively,the up and down axis may be generally described as the Z axis or Zdimension. The mechanical stage or arm may also include a hinge jointenabling a tilting motion or a ball joint enabling rotational motion. Ahinged joint enables an orifice of a transfer device to contact thebottom of a site at a slight angle so as to avoid the orifice fromsealing along the bottom of the site.

The transfer means may include at least one transfer device that has anorifice and a compartment capable of accommodating at least the greatestvolume of material required to be transferred in a single transfer stepand containing this volume during transfer from an origin site to adestination site. The compartment can be, but is not limited to, a tube,a chamber, a vessel, or a cavity. The transfer device may be selectedfrom, for example, a pipette, a needle, a tubing, a dropper, and thelike. The transfer device may be fitted with or otherwise include anozzle portion. In various embodiments the transfer means may include anarray of transfer devices and is capable of multi-channel transfer.Preferably, the number of transfer devices corresponds to the number ofconcurrent or sequential manipulation processes to be performed, e.g.,X. For example, each of a plurality of transfer devices is adapted tooperate on one among the plurality of strips.

The transfer device can be disposed on the robotic arm, thus it is movedto any desired sample site. Other devices that can move the transferdevice or the samples are also contemplated. For example, the transfermeans may include a motorized device to move the platform so that adesired sample can be moved to the transfer device, while the roboticarm can move in the Z axis to engage or disengage the transfer device.

The transfer means may further include a mechanism for acquiring ordischarging at least one volume of material. In various embodiments themechanism for acquiring or discharging a volume of material may be byvolume displacement. For example, the mechanism may be selected from apump, a suction bulb, a plunger, a syringe, a diaphragm, a piston andthe like. This mechanism may also be used as a sample treatment meansfor performing degassing, mixing, agitating, suspending particles andthe like. The volume transferred by the transfer means can be controlledby the apparatus control means. The volume may optionally be unique foreach transfer step.

In various embodiments the apparatus may further include a sampletreatment means for performing various treatment steps such asdegassing, mixing, heating, stirring and the like at a sample treatmentsite. The sample treatment means can comprise a transfer device andmechanism as described above in connection with the transfer means, aheating element such as a heating block or a heating coil, or a stirrersuch as a mechanical stirrer or a magnetic stirrer.

Various embodiments of the apparatus may include a separation means forseparating one or more samples at one or more separation sites. Invarious embodiments, the separation means may enable the separation of amulti-phase sample, e.g., a sample comprising a liquid fraction and asolid fraction of a sample at a separation site. In one embodiment, thesample comprises a liquid fraction and a solid fraction (e.g., solidparticles in a solution), at least a portion of the solid fraction (e.g.at least some of the solid particles) is magnetic and the separationmeans comprises a magnet. Examples of a magnetic solid fraction includemagnetic particles or beads. When the magnetic field of the magnet actson a separation site, the magnetic solid fraction of the sample issufficiently retained/held at the separation site so that thenon-magnetic solid or liquid fraction of the sample can be separatedfrom the magnetic solid fraction, for example by aspiration. Themagnetic field may be a persistent magnetic field such as that generatedby a permanent magnet, e.g., a bar magnet, or may be a temporarymagnetic field such as that generated by an electromagnet, i.e., anelectric current flowing through a conducting material such as a coiledwire e.g., a solenoid. In another embodiment, the sample comprises twoor more immiscible liquid fractions. The separation means separates thedifferent liquid fractions, e.g., by aspirating one liquid fraction. Inother embodiments, the sample comprises both two or more immiscibleliquid fractions and a magnetic solid fraction. The separation meansseparates these fractions by a combination of magnetic and liquidseparation discussed above.

In various embodiments, the apparatus may further include a positioningmeans for positioning various components of the apparatus. For example,the positioning means can bring various components of the apparatus intoan “IN” configuration and an “OUT” configuration. The IN and OUTconfigurations are characterized by whether the components can performtheir respective functions.

For example, in one embodiment in which the separation means comprises apermanent magnet, the positioning means is configured to move a sampleat a separation site or the magnet such that an IN configurationcorresponds to a configuration in which the sample is within themagnetic field of the magnet, whereas an OUT configuration correspondsto a configuration in which the sample is outside the magnetic field.

In an embodiment in which the separation means comprises a permanentmagnet, when the apparatus is in an IN configuration, the magnet is inproximity to a separation site such that the magnetic solid fraction atthe separation site will be retained during a separation step. When theapparatus is in an OUT configuration, the magnetic is not near theseparation site, i.e., the magnet is at a sufficient distance such thatthe magnetic solid fraction at the separation site will not be affectedby the magnetic field. The distance between a separation site and amagnet in the IN or OUT configuration may depend on the strength of themagnetic field or the nature of solid magnetic fraction in the sample. Aspatial relationships encompassing IN and OUT configurations is arrangedsuch that (1) the solid magnetic fraction is sufficiently retainedduring separation when the apparatus is in an IN configuration, and (2)the solid magnetic fraction is sufficiently unaffected by the magneticfield such that it may be manipulated, e.g., stirred, mixed, suspended,or transferred from the separation site when the apparatus is in an OUTconfiguration.

However, the positioning means does not need to actually move the sampleor the magnet. For example, in one embodiment in which the separationmeans comprises an electromagnet, the positioning means may beconfigured to turn on or off the magnetic field at a sample at aseparation site such that an IN configuration corresponds to aconfiguration that the magnetic field is on, whereas an OUTconfiguration corresponds to a configuration in which the magnetic fieldis off.

In another example, a positioning means can move various components ofthe apparatus into a configuration so that the treatment means can acton a sample at a sample treatment site.

Various embodiments of the apparatus may also include a capture meansadapted to capture one or more samples or fractions thereof at one ormore capture sites among said plurality of sites. The capture means mayinclude a capture magnet.

The positioning means may be an actuator, e.g., a stepper motor orsolenoid, which can engage various components of the apparatus to bemoved.

In various embodiments, the actuator can engage and move a magnet or anobject containing a magnet into an IN position and an OUT position andcan subsequently disengage.

In one embodiment, a rod extending from the transfer means can engageand move a magnet, or a block containing the magnet, into an INposition, i.e., placing the magnet adjacent to a separation site and canslide the block to an OUT position, i.e., placing magnet at a sufficientdistance so that the magnet is not affecting the magnetic solidfraction. The actuator can disengage at either the IN or the OUTposition so that various components of the apparatus are free to performother tasks. In such an embodiment, the rod and the transfer means serveas the actuator.

In alternative embodiments, the actuator can engage and move a sample ata separation site relative to a magnet and into an “IN” position and an“OUT” position and can subsequently disengage. For example, a rodextending from a transfer means can engage and move a separation siteinto an IN position, i.e., placing the separation site adjacent to amagnet and can position the separation site into an OUT position, i.e.,placing the separation site at a sufficient distance so that theseparation site is not sufficiently affected by the magnetic field. Theactuator can disengage so that various components of the apparatus arefree to perform other tasks.

In another embodiment, the actuator may be a rod extending from thetransfer means that engages and moves a sample into an IN position,i.e., placing the sample at a separation site adjacent to a magnet andcan slide the sample to an OUT position, i.e., placing the sample at aseparation site at a sufficient distance so that the magnet is notaffecting the magnetic solid fraction.

In a preferred embodiment, the transfer device also serves as theactuator. For example, the transfer device may be a pipette that servesas an actuator that engages and moves a sample at a separation sitetowards or away from a magnet, e.g., into an “IN” or an “OUT”configuration. Alternatively, the pipette may serve as an actuator thatengages and moves a magnet towards or away from a sample at a separationsite, e.g., into an “IN” or an “OUT” configuration.

Various embodiments of the present teachings may include a control meansfor controlling a transfer means, a separation means, a positioningmeans, and optionally a sample treatment means and capture means. Thecontrol means may receive instructions and cause the transfer means totransfer samples, the positioning means to relatively position theseparation means and sample, and the separation means to separate thesamples. The control means may include a user interface for allowing auser to set one or more operational parameters. The control means can beprogrammed to perform a variety of transferring, positioning, separatingand sample treatment steps on a plurality of samples without manualintervention. The control means may further include an electronicprocessing unit encoding various programs for controlling at least oneof the transfer means, separation means, positioning means, sampletreatment means and capture means.

An embodiment of the present teachings may include a computer programproduct, comprising a computer usable medium having a computer readableprogram encoded therein, the computer readable program adapted to beexecuted to implement a method using the apparatus according to thepresent teachings. The method comprises causing the transfer means totransfer one or more samples, causing the positioning means topositioning the separation means and one or more samples, causing theseparation means to separate one or more samples, and optionally causingthe sample treatment means to treat one or more samples or the capturemeans to capture one or more samples. In one embodiment, the computerprogram product further causes the apparatus to repeat these steps inany sequence without manual intervention.

Thus, an embodiment of the present teachings may include acomputer-readable storage medium having executable instructions forperforming the methods described above. The storage medium may be anytype of computer-readable medium (i.e., one capable of being read by acomputer), including non-transitory storage mediums such as magnetic oroptical tape or disks (e.g. hard disk or CD-ROM), solid state volatileor non-volatile memory, including random access memory (RAM), read-onlymemory (ROM), electronically programmable memory (EPROM or EEPROM), orflash memory. The term “non-transitory computer-readable storage medium”encompasses all computer-readable storage media, but excludes atransitory, propagating signal. As explained above, the instructions onthe computer-readable storage medium may control the operation of theapparatus of the present teachings.

Various embodiments may be implemented using hardware elements, softwareelements, or a combination of both. Examples of hardware elements mayinclude processors, microprocessors, circuits, circuit elements (e.g.,transistors, resistors, capacitors, inductors, and so forth), integratedcircuits, application specific integrated circuits (ASIC), programmablelogic devices (PLD), digital signal processors (DSP), field programmablegate array (FPGA), logic gates, registers, semiconductor device, chips,microchips, chip sets, and so forth. Examples of software may includesoftware components, programs, applications, computer programs,application programs, system programs, machine programs, operatingsystem software, middleware, firmware, software modules, routines,subroutines, functions, methods, procedures, software interfaces,application program interfaces (API), instruction sets, computing code,computer code, code segments, computer code segments, words, values,symbols, or any combination thereof. Determining whether an embodimentis implemented using hardware elements or software elements may vary inaccordance with any number of factors, such as desired computationalrate, power levels, heat tolerances, processing cycle budget, input datarates, output data rates, memory resources, data bus speeds and otherdesign or performance constraints.

Some embodiments may be implemented, for example, using acomputer-readable medium or article which may store an instruction or aset of instructions that, if executed by a machine, may cause themachine to perform a method or operations in accordance with theembodiments. Such a machine may include, for example, any suitableprocessing platform, computing platform, computing device, processingdevice, computing system, processing system, computer, processor, or thelike, and may be implemented using any suitable combination of hardwareor software. The computer-readable medium or article may include, forexample, any suitable type of memory unit, memory device, memoryarticle, memory medium, storage device, storage article, storage mediumor storage unit, for example, memory, removable or non-removable media,erasable or non-erasable media, writeable or re-writeable media, digitalor analog media, hard disk, floppy disk, read-only memory compact disc(CD-ROM), recordable compact disc (CD-R), rewriteable compact disc(CD-RW), optical disk, magnetic media, magneto-optical media, removablememory cards or disks, various types of Digital Versatile Disc (DVD), atape, a cassette, or the like. The instructions may include any suitabletype of code, such as source code, compiled code, interpreted code,executable code, static code, dynamic code, encrypted code, and thelike, implemented using any suitable high-level, low-level,object-oriented, visual, compiled or interpreted programming language.

Although described above separately, the present teachings contemplatethat one or more of the transfer means, separation means, positioningmeans, sample treatment means and capture means may be implemented byone or more shared devices. For example, the transfer means and thepositioning means may both comprise the same actuator which isconfigured to move the sample or the magnet.

The present teachings also provide a method for automated samplemanipulation of a plurality of samples. In one embodiment, the methodmay comprise providing the plurality of samples each at one of aplurality of sites, transferring one or more samples from one or moreorigin sites among to one or more destination sites, and separating eachof one or more samples at one or more separation sites into a pluralityof different fractions. The transferring and separating steps areperformed without manual intervention. In various embodiments, themethod may repeat the transferring and separating steps in any sequencea plurality of times.

In one embodiment, at least one of the plurality of samples comprises anonmagnetic portion and a magnetic portion, and the separating step maycomprise immobilizing the magnetic portion and removing the nonmagneticportion. In one embodiment, the nonmagnetic portion comprises a liquid,which may be a solution, and the magnetic portion comprises magneticparticles or beads. The separating step may comprise immobilizing themagnetic particles or beads by a magnet and removing the liquid byaspiration. In one embodiment, the magnetic beads comprise templatedbeads produced by PCR, e.g., by emulsion PCR. In one embodiment, themagnetic beads comprise magnetic enrichment beads, complexes of magneticenrichment bead and templated beads, and non-amplifying beads. Themethod may further comprise a step treating one or more samples at oneor more sample treatment sites. The treating step can comprise one ormore of degassing, mixing, heating, or stirring. The method may compriserepeating the steps transferring, separating, and treating (such as oneor more of degassing, mixing, heating, or stirring) in a predeterminedsequence to achieve the object of the automated process, e.g.,separation of the templated beads from other components.

In a preferred embodiment, the apparatus according to the presentteachings is for automated liquid sample manipulation, and includes aplatform holding eight 1×8 arrays, at least one magnet located near theend of the 1×8 array having a sample containing a magnetic portion to beseparated, a mechanical stage or arm having a mount for holding atransfer device, e.g., a pipette, a pump connected to the transferdevice and a control unit including a processer and memory encoding oneor more programs. The control unit is adapted to cause the mechanicalstage or arm and pump to carry out various tasks. The mechanical stageor arm is adapted to pick up the pipette; position the pipette at any ofthe plurality of sites and engage the transfer device with a liquidmaterial at that site. The mechanical stage or arm is also adapted touse the transfer device to move the 1×8 array on the platform so that asample is at a separation site such that the sample is within a magneticfield of said magnet.

In various embodiments of the present teachings, the method may becarried out as follows using an 8-well strip in which the user firstloads sample and all reagents (including wash solution) into the stripwells. The apparatus of the present teachings is then set to perform thefollowing sample manipulation sequence: 1) moving tip to well B andmixing solution via multiple aspirate/dispense cycles; 2) aspirating andtransferring solution to well A; 3) mixing solutions in well A viamultiple aspirate/dispense cycles; 4) actuating magnet, then aspiratingand transferring solution to well G; 5) de-actuating magnet; 6)aspirating wash solution from well C and transferring to well A; 7)mixing solutions in well A via multiple aspirate/dispense cycles; 8)actuating magnet, then aspirating and transferring solution back to wellC; 9) repeating wash cycle as in steps 6, 7 and 8 using wells D and E;10) de-actuating magnet and moving tip to well F,aspirating/transferring solution to well A; 11) mixing solutions in wellA via multiple aspirating/dispensing cycles; and 12) actuating magnetthen aspirating and transferring solution from well A to well H. Afterthe above steps, the user collects the processed sample in well H.

One embodiment of the automated sample handling device of the presentteachings is partially illustrated in FIGS. 1A, 1B and 1C. Theembodiment includes a platform 4 holding one X ×Y array 3, where (asshown) X is 1 and Y is 8, alternatively referred to as one eight sitestrip, preloaded with a magnetic fraction sample to be separated and anyreagents/solutions used in the automated process. The embodiment mayalso include an arm (e.g., a mechanical stage or arm) 11 holding apipette 2, connected to a pump (for example, pump 22 illustrated in FIG.3A) and an electronic control unit (for example, control unit 20illustrated in FIG. 3A) including a processer and memory encoding one ormore programs. The control unit is adapted to cause the mechanical stageor arm and pump to carry out various tasks. The mechanical stage or armis adapted to pick up the pipette; position the pipette at any of theplurality of sites and engage the transfer device with a liquid materialat that site. The mechanical stage or arm is also adapted to use thecontrol rod 8 to engage the slide block 7 to position the magnet 5 in anIN or an OUT (see, FIG. 1B, 7 a) position so that a sample is at aseparation site such that the sample is either within a magnetic fieldof the magnet or is sufficiently distant so as not to affect themagnetic fraction in the sample at the separation site. A second magnet6 is located at the opposite end of the 1 ×8 array and serves to captureany beads that may have been transferred to the last well. A rearperspective illustrated in FIG. 1 c shows holes 9 to accommodate controlrod while pipetting.

Another embodiment the automated sample handling device of the presentteachings is partially illustrated in FIG. 2. The embodiment is capableof multi-channel sample handling and includes a platform 4 holding a twodimensional array where each dimension is greater than 1, for example,an X×Y array of sites 3 where X is 8 and Y is 12. Alternatively, thearray of sites may be described as X of 1×Y strips, e.g., eight 1×12strips. The sites/strips may or may not be attached to each other. Eacharray or strip may be preloaded with samples containing a magneticfraction to be separated and any reagents/solutions used in theautomated process. The embodiment may also include an arm (e.g., amechanical stage or arm) 11 holding an array of pipette tips 2 eachconnected to a manifold 14 by tubing segments 9. The manifold 14 may beconnected to a pump and a control unit including a processer and memoryencoding one or more programs. Various embodiments may also includereagent bottles wherein the pump is adapted to deliver the contents ofthe reagent bottles to a plurality of sites. Alternatively, or inaddition, the embodiment may include a reservoir adapted receivecontents aspirated from at least one of a plurality of sites. Theembodiment may further include a bar magnet 5 that extends the Xdirection (as indicated in the figure) and spans all of the samplestrips. The bar magnet is positioned at either an IN position or an OUTposition by an actuator 15. A second magnet 6 (not pictured) mayoptionally be located at the opposite end of the 8×12 array as magnet 6and serves to capture any beads that may have been transferred to thelast well.

In various embodiments, the method according to the present teachingsmay be carried out as follows using a two dimensional array with X by Ynumber of sites, where X corresponds to the number of samples to bemanipulated by one automated process and where Y number of sites is 8.The apparatus according to the present embodiment includes an array oftips, e.g., for aspirating/dispensing/transferring, where the number oftips corresponds to the number of wells in the X dimension of the array.Embodiments of the automated sample handling device of the presentteachings such as the one partially illustrated in FIG. 2 may beparticularly well suited for performing the following method. Theapparatus may also include reagent bottles and a pump adapted to deliverthe contents of the reagent bottles to a plurality of sites. The userconnects all bottles and reservoirs, e.g., waste bottle and bottlescontaining wash solution and water. The user also loads samples andprimary reagents into wells and loads the array, which may be a plate orstrip. Then the apparatus of the present teachings is then set toperform the following sample manipulation sequence: 1) moving tip to Yposition B then mixing solution via multiple aspirate/dispense cycles,2) aspirating then transferring solution to Y position A wells, 3)mixing solutions at Y position A via multiple aspirate/dispense cycles4) actuating magnet, then aspirating and transferring solution to Yposition G wells (fail safe), 5) returning tip array to strip A, 6)de-actuating magnet, 7) dispensing wash solution and mix via multipleaspirate/dispense cycles, 8) actuating magnet then aspirating solutionto waste reservoir, 9) repeating wash cycle as in steps 6, 7 and 8, 10)moving tip Y position F and aspirating, then transferring solution to Yposition A wells, 11) de-actuating magnet and mixing solution viamultiple aspirate/dispense cycles. After the above steps, the usercollects the processed sample in Y position H wells.

In various embodiments of the present teachings, software may beprogrammed to execute the following commands: ‘Get’ which places themagnet in an IN position or turns the electromagnet on and ‘return’which places the magnet in an OUT position or turns the electromagnetoff; Go to Y position #; Aspirate×uL, where the user may input a volume,e.g., in an amount up to ˜300 uL in ˜10 uL increments; Dispense×uL,where the user may input a volume, e.g., in an amount up to ˜300 uL in˜10 uL increments; Aspirate/dispense speed, where the user may input aspeed e.g., ˜50 to 500 uL/sec in ˜50 uL/sec increments; Hold/delay wherethe user may input a time period, e.g., 0-150 seconds, 5 secondincrements. Variables available for user input also include parametersfor the mixing step: Number of cycles, Aspirate/dispense volumes,Aspirate speed, dispense speed, Hold/delay time between each cycle,Aspirate height, Dispense height. Volume accuracy: ˜5 uL. Generally, thefollowing variables are also set: Min volume/transfer: ˜100 uL, Maxvolume/transfer: ˜250 uL, Tip/nozzle positional accuracy: ˜1 mm in X &Y, ˜0.1 mm in Z, Magnet positional accuracy: ˜1 mm in X & Y and Z,Temporal accuracy: ˜1 second. The values may be adapted in accordancewith the requirements of any of a variety of sample handling proceduresand are not intended to be limiting. Other variables that considereduseful for any of a variety of procedures may also be included in thesoftware and available for input by the user.

Another embodiment of the automated sample handling device of thepresent teachings 1 is partially illustrated in FIGS. 3A, 3B and 3C. Theembodiment includes a platform 4 holding an X×Y array 3, where X is 1and Y is 8 (alternatively referred to as one eight site strip) preloadedwith a magnetic fraction sample to be separated and anyreagents/solutions/material used in the automated process. Theembodiment may also include an arm (e.g., a mechanical stage or arm) 11for holding a pipette 2, and a tubing port 12 for detachable connectionto a pump (not pictured) via tubing 9. The mechanical stage or arm maybe detachably connected to a cradle 10 and may form a hinged joint therebetween. The mechanical stage or arm may be adapted to use the pipetteto engage the array 3 to position the sample in an IN or an OUT positionsuch that the sample at a separation site is either within a magneticfield or is sufficiently distant so as not to affect the magneticfraction in the sample at the separation site, respectively.

The apparatus of the present teachings may further include a receptaclefor a container to which a processed sample may be transferred. Forexample, the container may be a tube (e.g., a PCR tube). The receptaclemay be placed at any suitable location in the apparatus as long as itcan be reached by the device mounted on the mechanical stage. In someembodiments, the receptacle and the container 17 is disposed next to theholder for holding the device (FIG. 3 c).

Another embodiment of the automated sample handling device of thepresent teachings is partially illustrated in FIG. 4. The embodimentincludes a platform 4 holding one X×Y array 3, where X is 1 and Y is 8,alternatively referred to as one eight site strip, preloaded with amagnetic fraction sample to be separated and anyreagents/solutions/material used in the automated process. Theembodiment may also include an arm (e.g., a mechanical stage or arm) 11for holding a pipette 2, and has a tubing port 13 for detachableconnection to a pump (not pictured) via tubing 9. The mechanical stageor arm may be detachably connected to a cradle 10 and may form a hingedjoint there between. The mechanical stage or arm may be adapted to usethe control rod 8 to engage the slide block 7 to position the magnet 5in an IN or an OUT position where the sample at a separation site iseither within a magnetic field or is sufficiently distant so as not toaffect the magnetic fraction in the sample at the separation site,respectively. Alternatively or additionally, an actuator or solenoid(not pictured) may be used to position the magnet 5 in an IN or OUTposition. A second magnet 6 (not pictured) may be located at theopposite end of the array and serves to capture any beads that may havebeen transferred to the last well.

Another embodiment of the automated sample handling device of thepresent teachings is partially illustrated in FIG. 5. The embodimentincludes a platform 4 holding one X×Y array 3, where X is 1 and Y is 8,alternatively referred to as one eight site strip, which may bepreloaded with a magnetic fraction sample to be separated and anyreagents/solutions/material used in the automated process. Theembodiment may also include an arm (e.g., a mechanical stage or arm) 11for holding a pipette 2, and may optionally have a tubing port 13 fordetachable connection to a pump via tubing 9. Alternatively, the pipettemay be connected to an automatic volumetric pipette operated by a pistonor the like. The mechanical stage or arm may be detachably connected toa cradle 10 and may form a hinged joint there between. The mechanicalstage or arm may be adapted to position the pipette at any of theplurality of sites and engage the transfer device with a liquid materialat that site. The mechanical stage or arm may further be adapted to pickup the pipette. An actuator or solenoid may be used to position themagnet 5 in an IN or an OUT position where the sample at a separationsite is either within a magnetic field or is sufficiently distant so asnot to affect the magnetic fraction in the sample at the separationsite, respectively. A second magnet 6 (not pictured) may be located atthe opposite end of the array and serves to capture any beads that mayhave been transferred to the last well.

In a first aspect, an apparatus for automated liquid sample manipulationincludes an array of compartments, a magnet, a translation deviceincluding a mounting for a pipette tip, and a control unit to facilitaterelative movement of the array and the magnet to apply and remove amagnetic field from at least one compartment of the array. The controlunit is to control the translation device to position the pipette tip inthe at least one compartment.

In an example of the first aspect, the magnet is movable, the controlunit to facilitate movement of the magnet into and out of proximity tothe at least one compartment of the array. For example, the control unitis to control the translation device to move the magnet. In an example,the translation device includes a rod and the magnet is disposed withina movable sled configured to receive the rod. In a further example, themagnet is disposed within a movable sled having a rod, the translationto engage the rod to move the sled.

In another example of the first aspect and the above examples, themagnet is stationary and the array of compartments is movable, thecontrol unit to control the translation device to move the at least onecompartment into proximity with the magnet. For example, the apparatusfurther includes a platform including a channel to receive the array ofcompartments, the array of compartments movable along the channel.

In a further example of the first aspect and the above examples, theapparatus includes a pump in communication with the pipette tip tofacilitate aspiration and deposition of fluids.

In an additional example of the first aspect and the above examples, thearray of compartments is a strip of tubes.

In another example of the first aspect and the above examples, the arrayof compartments is a tray of wells.

In a second aspect, an apparatus includes a platform to receive an arrayof compartments, a magnet in proximity to the platform, a translationdevice including a mounting for a pipette tip, and a control unit tofacilitate relative movement of the array and the magnet to apply andremove a magnetic field from at least one compartment of the array. Thecontrol unit is to control the translation device to position thepipette tip in the at least one compartment.

In an example of the second aspect, the magnet is movable, the controlunit to facilitate movement of the magnet into and out of proximity tothe at least one compartment of the array. For example, the control unitis to control the translation device to move the magnet. In anotherexample, the translation device includes a rod and the magnet isdisposed within a movable sled configured to receive the rod. In anadditional example, the magnet is disposed within a movable sled havinga rod, the translation to engage the rod to move the sled.

In another example of the second aspect and the above examples, themagnet is stationary and the array of compartments is movable relativeto the platform, the control unit to control the translation device tomove the at least one compartment into proximity with the magnet. Forexample, the platform includes a channel to receive the array ofcompartments, the array of compartments movable along the channel.

In a further example of the second aspect and the above examples, theapparatus further includes a pump in communication with the pipette tipto facilitate aspiration and deposition of fluids.

In an additional example of the second aspect and the above examples,the array of compartments is a strip of tubes.

In another example of the second aspect and the above examples, thearray of compartments is a tray of wells.

In a third aspect, a method for enriching templated beads includesforming a complex including a magnetic enrichement bead and a templatedbead, applying a magnetic field to the complex to secure the complex,washing the complex, and separating the templated bead from the magneticenrichment bead.

In an example of the third aspect, separating the template bead from themagnetic enrichment bead includes breaking the complex into a separatemagnetic enrichment bead and a template bead, applying a magnetic fieldto secure the separate magnetic enrichment bead, and removing thetemplate bead.

In another example of the third aspect and the above examples, applyingthe magnetic field includes moving a magnet into position adjacent atube including the complex.

In an additional example of the third aspect and the above examples,applying the magnetic field includes moving a tube including the complexinto position adjacent a magnet.

In a further example of the third aspect and the above examples,applying the magnetic field includes activating an electromagnetadjacent a tube including the complex.

In a fourth aspect, a method of enriching templated beads includesmixing a dispersion of templated beads with magnetic enrichment beads ina well to form complexes including at least one templated bead and atleast one magnetic enrichment bead, applying a magnetic field to thecontainer to secure the complexes to a wall of the well, washing thewell with a wash solution, and removing the magnetic field.

In an example of the fourth aspect, the method further includesseparating the templated beads from the complexes, applying a magneticfield to secure the magnetic enrichment beads, and removing thetemplated beads from the well.

In another example of the fourth aspect and the above examples, applyingthe magnetic field includes moving a magnet into position adjacent thewell including the complexes.

In a further example of the fourth aspect and the above examples,applying the magnetic field includes moving the well including thecomplexes into position adjacent a magnet.

In an additional example of the fourth aspect and the above examples,applying the magnetic field includes activating an electromagnetadjacent the well including the complexes.

For the purposes of this specification and appended claims, unlessotherwise indicated, all numbers expressing quantities, percentages orproportions, and other numerical values used in the specification andclaims, are to be understood as being modified in all instances by theterm “about” or the symbol “˜.” Accordingly, unless indicated to thecontrary, the numerical parameters set forth in the followingspecification and attached claims are approximations that may varydepending upon the desired properties sought to be obtained by thepresent invention. At the very least, and not as an attempt to limit theapplication of the doctrine of equivalents to the scope of the claims,each numerical parameter should at least be construed in light of thenumber of reported significant digits and by applying ordinary roundingtechniques.

Note that not all of the activities described above in the generaldescription or the examples are required, that a portion of a specificactivity may not be required, and that one or more further activitiesmay be performed in addition to those described. Still further, theorder in which activities are listed are not necessarily the order inwhich they are performed.

In the foregoing specification, the concepts have been described withreference to specific embodiments. However, one of ordinary skill in theart appreciates that various modifications and changes can be madewithout departing from the scope of the invention as set forth in theclaims below. Accordingly, the specification and figures are to beregarded in an illustrative rather than a restrictive sense, and allsuch modifications are intended to be included within the scope ofinvention.

As used herein, the terms “comprises,” “comprising,” “includes,”“including,” “has,” “having” or any other variation thereof, areintended to cover a non-exclusive inclusion. For example, a process,method, article, or apparatus that comprises a list of features is notnecessarily limited only to those features but may include otherfeatures not expressly listed or inherent to such process, method,article, or apparatus. Further, unless expressly stated to the contrary,“or” refers to an inclusive-or and not to an exclusive-or. For example,a condition A or B is satisfied by any one of the following: A is true(or present) and B is false (or not present), A is false (or notpresent) and B is true (or present), and both A and B are true (orpresent).

Also, the use of “a” or “an” are employed to describe elements andcomponents described herein. This is done merely for convenience and togive a general sense of the scope of the invention. This descriptionshould be read to include one or at least one and the singular alsoincludes the plural unless it is obvious that it is meant otherwise.

Benefits, other advantages, and solutions to problems have beendescribed above with regard to specific embodiments. However, thebenefits, advantages, solutions to problems, and any feature(s) that maycause any benefit, advantage, or solution to occur or become morepronounced are not to be construed as a critical, required, or essentialfeature of any or all the claims.

After reading the specification, skilled artisans will appreciate thatcertain features are, for clarity, described herein in the context ofseparate embodiments, may also be provided in combination in a singleembodiment. Conversely, various features that are, for brevity,described in the context of a single embodiment, may also be providedseparately or in any subcombination. Further, references to valuesstated in ranges include each and every value within that range.

What is claimed is:
 1. An apparatus for automated liquid samplemanipulation, the apparatus comprising: a platform including ahorizontal surface; an array of compartments disposed in a first portionof a channel of the platform and extending in the horizontal surface: amagnet disposed in a second portion of the channel of the platformextending in the horizontal surface, the array of compartments and themagnet movable relative to each other over the horizontal surface; atranslation device including a mounting for a pipette tip the pipettetip mounted to the mounting of the translation device; and an electroniccontrol unit programmed to facilitate relative movement of the array andthe magnet along the respective first or second channels to apply andremove a magnetic field from at least one compartment of the array, theelectronic control unit programmed to control the translation device toposition the pipette tip in the at least one compartment.
 2. Theapparatus of claim 1, wherein the magnet is movable, the electroniccontrol unit programmed to facilitate movement of the magnet to applyand remove the magnetic field from the at least one compartment of thearray.
 3. The apparatus of claim 2, wherein the electronic control unitis programmed to control the translation device to move the magnet. 4.The apparatus of claim 3, wherein the translation device includes a rodand the magnet is disposed within a movable sled configured to receivethe rod.
 5. The apparatus of claim 3, wherein the magnet is disposedwithin a movable sled within the second channel, the movable sled havinga rod, the electronic control unit programmed to control the translationdevice to engage the rod to move the sled.
 6. The apparatus of claim 1,wherein the magnet is stationary and the array of compartments ismovable along the first channel, the electronic control unit programmedto control the translation device to move the at least one compartmenttoward the magnet to apply the magnetic field.
 7. The apparatus of claim6, wherein the electronic control unit is programmed to control thetranslation device to move the array of compartments using the pipettetip.
 8. The apparatus of claim 1, further comprising a pump in fluidcommunication with the pipette tip to facilitate aspiration anddeposition of fluids from compartments of the array of compartments. 9.The apparatus of claim 1, wherein the array of compartments is a stripof tubes.
 10. The apparatus of claim 1, wherein the array ofcompartments is a tray of wells.
 11. An apparatus comprising: a platformto receive including a channel to receive an array of compartments thechannel extending in a horizontal surface of the platform; the array ofcompartments disposed in the channel; a magnet disposed in thehorizontal surface of the platform and proximal to an end compartment ofthe array of compartments; a translation device including a mounting fora pipette tip the pipette tip mounted to the mounting of the translationdevice; and an electronic control unit programmed to facilitate relativemovement of the array and the magnet to apply and remove a magneticfield from at least one compartment of the array, the electronic controlunit programmed to control the translation device to position thepipette tip in the at least one compartment.
 12. The apparatus of claim11, wherein the magnet is movable relative to the platform, theelectronic control unit programmed to facilitate movement of the magnetto apply a magnetic field to or remove the magnetic field from the atleast one compartment of the array.
 13. The apparatus of claim 12,wherein the electronic control unit is programmed to control thetranslation device to move the magnet.
 14. The apparatus of claim 13,wherein the translation device includes a rod and the magnet is disposedwithin a movable sled configured to receive the rod.
 15. The apparatusof claim 13, wherein the magnet is disposed within a movable sled havinga rod, the electronic control unit programmed to control the translationdevice to engage the rod to move the sled.
 16. The apparatus of claim11, wherein the magnet is stationary and the array of compartments ismovable along the channel relative to the platform, the electroniccontrol unit programmed to control the translation device to move the atleast one compartment into proximity with the magnet.
 17. The apparatusof claim 16, wherein the electronic control unit is programmed tocontrol the translation device to move the array of compartments usingthe pipette tip.
 18. The apparatus of claim 11, further comprising apump in fluid communication with the pipette tip to facilitateaspiration and deposition of fluids.
 19. The apparatus of claim 11,further comprising the array of compartments, wherein the array ofcompartments is a strip of tubes.